Polynucleotides encoding a multiple epitope fusion antigen for use in an HCV antigen/antibody combination assay

ABSTRACT

The invention relates to a method of detecting HCV infection in a biological sample, the method comprising providing an immunoassay solid support, comprising an HCV anti-core antibody, an antigen comprising an HCV NS3/4 a  epitope, and an HCV multiple epitope fusion antigen, that can detect both HCV antigens and antibodies present in a sample. The invention also includes polynucleotides encoding multiple epitope fusion antigens for use in the assay, recombinant vectors and host cells comprising such polynucleotides, and methods of producing the multiple epitope fusion antigens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/881,239, filed Jun. 14, 2001, now U.S. Pat. No. 6,630,298 which isrelated to provisional patent application Ser. Nos. 60/212,082, filedJun. 15, 2000; 60/280,867, filed Apr. 2, 2001; and 60/280,811, filedApr. 2, 2001, from which applications priority is claimed under 35 USC§§ 120 and 119(e)(1) and which applications are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present invention pertains generally to viral diagnostics. Inparticular, the invention relates to an antigen/antibody combinationassay for accurately diagnosing hepatitis C virus infection.

BACKGROUND OF THE INVENTION

Hepatitis C Virus (HCV) is the principal cause of parenteral non-A,non-B hepatitis (NANBH) which is transmitted largely through bloodtransfusion and sexual contact. The virus is present in 0.4 to 2.0% ofblood donors. Chronic hepatitis develops in about 50% of infections andof these, approximately 20% of infected individuals develop livercirrhosis which sometimes leads to hepatocellular carcinoma.Accordingly, the study and control of the disease is of medicalimportance.

HCV was first identified and characterized as a cause of NANBH byHoughten et al. The viral genomic sequence of HCV is known, as aremethods for obtaining the sequence. See, e.g., International PublicationNos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kbpositive-sense, single-stranded RNA genome and is a member of theFlaviridae family of viruses. At least six distinct, but relatedgenotypes of HCV, based on phylogenetic analyses, have been identified(Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodesa single polyprotein having more than 3000 amino acid residues (Choo etal., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA(1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991)88:1711-1715). The polyprotein is processed co- and post-translationallyinto both structural and non-structural (NS) proteins.

In particular, as shown in FIG. 1, several proteins are encoded by theHCV genome. The order and nomenclature of the cleavage products of theHCV polyprotein is as follows:NH₂-C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial cleavage of thepolyprotein is catalyzed by host proteases which liberate threestructural proteins, the N-terminal nucleocapsid protein (termed “core”)and two envelope glycoproteins, “E1” (also known as E) and “E2” (alsoknown as E2/NS1), as well as nonstructural (NS) proteins that containthe viral enzymes. The NS regions are termed NS2, NS3, NS4 and NS5. NS2is an integral membrane protein with proteolytic activity. NS2, eitheralone or in combination with NS3, cleaves the NS2-NS3 sissle bond whichin turn generates the NS3 N-terminus and releases a large polyproteinthat includes both serine protease and RNA helicase activities. The NS3protease serves to process the remaining polyprotein. Completion ofpolyprotein maturation is initiated by autocatalytic cleavage at theNS3-NS4a junction, catalyzed by the NS3 serine protease. SubsequentNS3-mediated cleavages of the HCV polyprotein appear to involverecognition of polyprotein cleavage junctions by an NS3 molecule ofanother polypeptide. In these reactions, NS3 liberates an NS3 cofactor(NS4a), two proteins (NS4b and NS5a), and an RNA-dependent RNApolymerase (NS5b).

A number of general and specific polypeptides useful as immunologicaland diagnostic reagents for HCV, derived from the HCV polyprotein, havebeen described. See, e.g., Houghton et al., European Publication Nos.318,216 and 388,232; Choo et al., Science (1989) 244:359-362; Kuo etal., Science (1989) 244:362-364; Houghton et al., Hepatology (1991)14:381-388; Chien et al., Proc. Natl. Acad. Sci. USA (1992)89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39;Chien et al., International Publication No. WO 93/00365; Chien, D. Y.,International Publication No. WO 94/01778. These publications provide anextensive background on HCV generally, as well as on the manufacture anduses of HCV polypeptide immunological reagents. For brevity, therefore,the disclosure of these publications is incorporated herein byreference.

Sensitive, specific methods for screening and identifying carriers ofHCV and HCV-contaminated blood or blood products would provide animportant advance in medicine. Post-transfusion hepatitis (PTH) occursin approximately 10% of transfused patients, and HCV has accounted forup to 90% of these cases. Patient care as well as the prevention andtransmission of HCV by blood and blood products or by close personalcontact require reliable diagnostic and prognostic tools. Accordingly,several assays have been developed for the serodiagnosis of HCVinfection. See, e.g., Choo et al., Science (1989) 244:359-362; Kuo etal., Science (1989) 244:362-364; Choo et al., Br. Med. Bull. (1990)46:423-441; Ebeling et al., Lancet (1990) 335:982-983; van der Poel etal., Lancet (1990) 335:558-560; van der Poel et al., Lancet (1991)337:317-319; Chien, D. Y., International Publication No. WO 94/01778;Valenzuela et al., International Publication No. WO 97/44469; andKashiwakuma et al., U.S. Pat. No. 5,871,904.

A significant problem encountered with some serum-based assays is thatthere is a significant gap between infection and detection of the virus,often exceeding 80 days. This assay gap may create great risk for bloodtransfusion recipients. To overcome this problem, nucleic acid-basedtests (NAT) that detect viral RNA directly, and HCV core antigen teststhat assay viral antigen instead of antibody response, have beendeveloped. See, e.g., Kashiwakuma et al., U.S. Pat. No. 5,871,904; Beldet al., Transfusion (2000) 40:575-579.

However, there remains a need for sensitive, accurate diagnostic andprognostic tools in order to provide adequate patient care as well as toprevent transmission of HCV by blood and blood products or by closepersonal contact.

SUMMARY OF THE INVENTION

The present invention is based in part, on the finding that HCVseroconversion antibodies are typically anti-core and anti-NS3(helicase). Accordingly, the invention provides an HCV core antigen andNS3 antibody combination assay that can detect both HCV antigens andantibodies present in a sample using a single solid matrix.

Accordingly, in one embodiment, the subject invention is directed to animmunoassay solid support comprising at least one HCV anti-core antibodyand at least one isolated HCV NS3/4a epitope bound thereto. The antibodyand NS3/4a epitope can be any of the herein described molecules.Additionally, the solid support may include any of the multiple epitopefusion antigens described herein, such as the multiple epitope fusionantigen comprising the amino acid sequence depicted in FIGS. 7A-7F.

In certain embodiments, the solid support comprises at least two HCVanti-core antibodies bound thereto. Moreover, the anti-core antibody maybe a monoclonal antibody. Additionally, the NS3/4a epitope may be aconformational epitope, such as a conformational NS3/4a epitopecomprising the amino acid sequence depicted in FIGS. 4A-4D.

In another embodiment, the invention is directed to an imununoassaysolid support comprising at least two HCV anti-core monoclonalantibodies and at least one HCV NS3/4a conformational epitope comprisingthe amino acid sequence depicted in FIGS. 4A-4D, bound thereto.

In still a further embodiment, the invention is directed to a method ofdetecting HCV infection in a biological sample. The method comprises:(a) providing an immunoassay solid support as described above; (b)combining a biological sample with the solid support under conditionswhich allow HCV antigens and antibodies, when present in the biologicalsample, to bind to the at least one anti-core antibody and the NS3/4aepitope, respectively; (c) adding to the solid support from step (b)under complex forming conditions (i) a first detectably labeledantibody, wherein the first detectably labeled antibody is a detectablylabeled HCV anti-core antibody, wherein the labeled anti-core antibodyis directed against a different HCV core epitope than the at least oneanti-core antibody bound to the solid support; (ii) an antigen thatreacts with an HCV antibody from the biological sample reactive with theNS3/4a epitope; and (iii) a second detectably labeled antibody, whereinthe second detectably labeled antibody is reactive with the antigen of(ii); and (d) detecting complexes formed between the antibodies andantigens, if any, as an indication of HCV infection in the biologicalsample. The NS3/4a epitope may be a conformational epitope, such as aconformational epitope having the NS3/4a sequence depicted in FIGS.4A-4D.

In yet another embodiment, the invention is directed to a method ofdetecting HCV infection in a biological sample. The method comprises:(a) providing an immunoassay solid support with at least two HCVanti-core antibodies bound thereto, as described above; (b) combining abiological sample with the solid support under conditions which allowHCV antigens and antibodies, when present in the biological sample, tobind to the at least two anti-core antibodies and the NS3/4a epitope,respectively; (c) adding to the solid support from step (b) undercomplex forming conditions (i) a first detectably labeled antibody,wherein the first detectably labeled antibody is a detectably labeledHCV anti-core antibody, wherein the labeled anti-core antibody isdirected against a different HCV core epitope than the anti-coreantibodies bound to the solid support; (ii) an epitope from the c33cregion of the HCV polyprotein fused to an hSOD amino acid sequence; and(iii) a second detectably labeled antibody, wherein the seconddetectably labeled antibody is reactive with the hSOD amino acidsequence; and (d) detecting complexes formed between the antibodies andantigens, if any, as an indication of HCV infection in the biologicalsample. The NS3/4a epitope may be a conformational epitope, such as aconformational epitope having the NS3/4a sequence depicted in FIGS.4A-4D.

In any of the above embodiments, the anti-core antibody may be directedagainst an N-terminal region of the HCV core antigen, such as againstamino acids 10-53 of HCV, numbered relative to the HCV1 polyproteinsequence, and/or the detectably labeled HCV anti-core antibody may bedirected against a C-terminal region of the HCV core antigen, such asamino acids 120-130 of HCV, numbered relative to the HCV1 polyproteinsequence. Moreover, the antigen that reacts with an HCV antibody fromthe biological sample may be from the NS3 region, such as an epitopefrom the c33c region of the HCV polyprotein and can be fused with ahuman superoxide dismutase (hSOD) amino acid sequence. In thisembodiment, the second detectably labeled antibody is reactive with thehSOD amino acid sequence.

In another embodiment, the invention is directed to a method ofdetecting HCV infection in a biological sample. The method comprises:(a) providing an immunoassay solid support including two HCV anti-coremonoclonal antibodies and a conformational epitope comprising the aminoacid sequence depicted in FIGS. 4A-4D; (b) combining a biological samplewith the solid support under conditions which allow HCV antigens andantibodies, when present in the biological sample, to bind to the atleast two anti-core antibodies and the NS3/4a conformational epitope,respectively; adding to the solid support from step (b) under complexforming conditions (i) a first detectably labeled antibody, wherein thefirst detectably labeled antibody is a detectably labeled HCV anti-coreantibody, wherein the labeled anti-core antibody is directed against adifferent HCV core epitope than the at least two anti-core antibodiesbound to the solid support; (ii) an epitope from the c33c region of theHCV polyprotein fused to an hSOD amino acid sequence; and (iii) a seconddetectably labeled antibody, wherein the second detectably labeledantibody is reactive with said hSOD amino acid sequence; detectingcomplexes formed between the antibodies and antigens, if any, as anindication of HCV infection in the biological sample.

In certain embodiments, the at least two anti-core antibodies aredirected against an N-terminal region of the HCV core antigen, such asagainst amino acids 10-53 of HCV, numbered relative to the HCV1polyprotein, and the detectably labeled HCV anti-core antibody isdirected against a C-terminal region of the HCV core antigen, such asagainst amino acids 120-130 of HCV, numbered relative to the HCVpolyprotein sequence.

In another embodiment, the invention is directed to a method ofdetecting HCV infection in a biological sample. The method comprises:(a) providing an immunoassay solid support which includes a multipleepitope fusion antigen; (b) combining a biological sample with the solidsupport under conditions which allow HCV antigens and antibodies, whenpresent in the biological sample, to bind to the at least one anti-coreantibody, the NS3/4a epitope, and the multiple epitope fusion antigen;(c) adding to the solid support from step (b) under complex formingconditions (i) a first detectably labeled antibody, wherein the firstdetectably labeled antibody is a detectably labeled HCV anti-coreantibody, wherein the labeled anti-core antibody is directed against adifferent HCV core epitope than the at least one anti-core antibodybound to the solid support; (ii) first and second antigens that reactwith an HCV antibody from the biological sample reactive with the NS3/4aepitope and the multiple epitope fusion antigen, respectively; and (iii)a second detectably labeled antibody, wherein the second detectablylabeled antibody is reactive with the antigens of (ii); (d) detectingcomplexes formed between the antibodies and antigens, if any, as anindication of HCV infection in the biological sample.

The anti-core antibody may be directed against an N-terminal region ofthe HCV core antigen and said first detectably labeled HCV anti-coreantibody may be directed against a C-terminal region of the HCV coreantigen, as described above. Moreover, the first antigen that reactswith an HCV antibody from the biological sample may comprise an epitopefrom the c33c region of the HCV polyprotein, and may be fused with anhSOD amino acid sequence. In this context, the second detectably labeledantibody is reactive with the hSOD amino acid sequence. Additionally,the second antigen that reacts with an HCV antibody from the biologicalsample may comprise an epitope from the c22 region of the HCVpolyprotein, such as an epitope comprising amino acids Lys₁₀ to Ser₉₉ ofthe HCV polyprotein, with a deletion of Arg47 and a substitution of Leufor Trp at position 44, numbered relative to the HCV1 polyproteinsequence. The epitope may be fused with an hSOD amino acid sequence. Ifso, the second detectably labeled antibody is reactive with the hSODamino acid sequence. The multiple epitope fusion antigen may comprisethe amino acid sequence depicted in FIGS. 7A-7F.

In yet a further embodiment, the invention is directed to a method ofdetecting HCV infection in a biological sample, said method comprising:(a) providing an immunoassay solid support which comprises two HCVanti-core monoclonal antibodies, an HCV NS3/4a conformational epitopecomprising the amino acid sequence depicted in FIGS. 4A-4D, and amultiple epitope fusion antigen comprising the amino acid sequencedepicted in FIGS. 7A-7F, bound thereto; (b) combining a biologicalsample with the solid support under conditions which allow HCV antigensand antibodies, when present in the biological sample, to bind to the atleast two anti-core antibodies, the NS3/4a conformational epitope, andthe multiple epitope fusion antigen, respectively; (c) adding to thesolid support from step (b) under complex forming conditions (i) a firstdetectably labeled antibody, wherein the first detectably labeledantibody is a detectably labeled HCV anti-core antibody, wherein thelabeled anti-core antibody is directed against a different HCV coreepitope than the at least two anti-core antibodies bound to the solidsupport; (ii) an epitope from the c33c region of the HCV polyproteinfused to an hSOD amino acid sequence and an epitope from the c22 regionof the HCV polyprotein fused to an hSOD amino acid sequence; and (iii) asecond detectably labeled antibody, wherein said second detectablylabeled antibody is reactive with said hSOD amino acid sequences; (d)detecting complexes formed between the antibodies and antigens, if any,as an indication of HCV infection in the biological sample.

In this embodiment, the at least two anti-core antibodies may bedirected against an N-terminal region of the HCV core antigen, such asagainst amino acids 10-53 of HCV, numbered relative to the HCV1polyprotein, and the detectably labeled HCV anti-core antibody isdirected against a C-terminal region of the HCV core antigen, such asagainst amino acids 120-130 of HCV, numbered relative to the HCV1polyprotein sequence. Moreover, the epitope from the c22 region maycomprise amino acids Lys₁₀ to Ser₉₉ of the HCV polyprotein, with adeletion of Arg47 and a substitution of Leu for Trp at position 44,numbered relative to the HCV1 polyprotein sequence.

In other embodiments, the invention is directed to immunodiagnostic testkits comprising the immunoassay solid support described above, andinstructions for conducting the immunodiagnostic test.

In still further embodiments, the invention is directed to methods ofproducing an immunoassay solid support, comprising: (a) providing asolid support; and (b) binding at least one HCV anti-core antibody, suchas one or two or more, and at least one isolated HCV NS3/4a epitopethereto, and optionally, a multiple epitope fusion antigen thereto. Theanti-core antibodies, NS3/4a epitopes and multiple epitope fusionantigens are as described above.

In additional embodiments, the invention is directed to a multipleepitope fusion antigen comprising the amino acid sequence depicted inFIGS. 7A-7F, or an amino acid sequence with at least 80% sequenceidentity, such as 90% or more sequence identity, thereto which reactsspecifically with anti-HCV antibodies present in a biological samplefrom an HCV-infected individual. In certain embodiments, the multipleepitope fusion antigen consists of the amino acid sequence depicted inFIGS. 5A-5F.

In further embodiments, the invention is directed to a polynucleotidecomprising a coding sequence for the multiple epitope fusion antigenabove, a recombinant vectors comprising the polynucleotides, host cellstransformed with the recombinant vectors, and methods of producing arecombinant multiple epitope fusion antigen comprising: (a) providing apopulation of host cells as above; and (b) culturing the population ofcells under conditions whereby the multiple epitope fusion antigenencoded by the coding sequence present in the recombinant vector isexpressed.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions, and aretherefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the HCV genome, depicting thevarious regions of the polyprotein from which the present assay reagents(proteins and antibodies) are derived.

FIG. 2 is a schematic drawing of a representative antibody/antigencombination assay under the invention.

FIG. 3 (SEQ ID NO:1) depicts the amino acid sequence of a representativeNS3/4a conformational antigen for use in the present assays. The boldedalanine at position 182 is substituted for the native serine normallypresent at this position.

FIGS. 4A through 4D depict the DNA (SEQ ID NO:2) and corresponding aminoacid (SEQ ID NO:3) sequence of another representative NS3/4aconformational antigen for use in the present assays. The amino acids atpositions 403 and 404 of FIGS. 4A through 4D represent substitutions ofPro for Thr, and Ile for Ser, of the native amino acid sequence ofHCV-1.

FIG. 5 is a diagram of the construction of pd.HCV1a.ns3ns4aPI.

FIG. 6 is a diagrammatic representation of MEFA 12.

FIGS. 7A-7F depict the DNA (SEQ ID NO:4) and corresponding amino acid(SEQ ID NO:5) sequence of MEFA 12.

FIG. 8 is a schematic drawing of a representative immunoassay under theinvention, using MEFA 12.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, recombinantDNA techniques and immunology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., FundamentalVirology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.);Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., Blackwell Scientific Publications); T. E. Creighton,Proteins: Structures and Molecular Properties (W.H. Freeman and Company,1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., currentaddition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2ndEdition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds.,Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “an antigen” includes a mixture of two or more antigens,and the like.

The following amino acid abbreviations are used throughout the text:

Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid:Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E)Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L)Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro(P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr(Y) Valine: Val (V)

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the proteins or errorsdue to PCR amplification.

An HCV polypeptide is a polypeptide, as defined above, derived from theHCV polyprotein. The polypeptide need not be physically derived fromHCV, but may be synthetically or recombinantly produced. Moreover, thepolypeptide may be derived from any of the various HCV strains, such asfrom strains 1, 2, 3 or 4 of HCV. A number of conserved and variableregions are known between these strains and, in general, the amino acidsequences of epitopes derived from these regions will have a high degreeof sequence homology, e.g., amino acid sequence homology of more than30%, preferably more than 40%, when the two sequences are aligned. Thus,for example, the term “NS3/4a” polypeptide refers to native NS3/4a fromany of the various HCV strains, as well as NS3/4a analogs, muteins andimmunogenic fragments, as defined further below. The complete genotypesof many of these strains are known. See, e.g., U.S. Pat. No. 6,150,087and GenBank Accession Nos. AJ238800 and AJ238799.

The terms “analog” and “mutein” refer to biologically active derivativesof the reference molecule, or fragments of such derivatives, that retaindesired activity, such as immunoreactivity in the assays describedherein. In general, the term “analog” refers to compounds having anative polypeptide sequence and structure with one or more amino acidadditions, substitutions (generally conservative in nature) and/ordeletions, relative to the native molecule, so long as the modificationsdo not destroy immunogenic activity. The term “mutein” refers topeptides having one or more peptide mimics (“peptoids”), such as thosedescribed in International Publication No. WO 91/04282. Preferably, theanalog or mutein has at least the same immunoactivity as the nativemolecule. Methods for making polypeptide analogs and muteins are knownin the art and are described further below.

Particularly preferred analogs include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

By “fragment” is intended a polypeptide consisting of only a part of theintact full-length polypeptide sequence and structure. The fragment caninclude a C-terminal deletion and/or an N-terminal deletion of thenative polypeptide. An “immunogenic fragment” of a particular HCVprotein will generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, that define an epitope, or any integerbetween 5 amino acids and the full-length sequence, provided that thefragment in question retains immunoreactivity in the assays describedherein. For example, preferred immunogenic fragments, include but arenot limited to fragments of HCV core that comprise, e.g., amino acids10-45, 10-53, 67-88, and 120-130 of the polyprotein, epitope 5-1-1 (inthe NS3 region of the viral genome) as well as defined epitopes derivedfrom the E1, E2, c33c (NS3), c100 (NS4), NS3/4a and NS5 regions of theHCV polyprotein, as well as any of the other various epitopes identifiedfrom the HCV polyprotein. See, e.g., Chien et al., Proc. Natl. Acad.Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol.(1993) 8:S33-39; Chien et al., International Publication No. WO93/00365; Chien, D. Y., International Publication No. WO 94/01778; U.S.Pat. Nos. 6,150,087 and 6,121,020, all of which are incorporated byreference herein.

The term “epitope” as used herein refers to a sequence of at least about3 to 5, preferably about 5 to 10 or 15, and not more than about 1,000amino acids (or any integer therebetween), which define a sequence thatby itself or as part of a larger sequence, binds to an antibodygenerated in response to such sequence. There is no critical upper limitto the length of the fragment, which may comprise nearly the full-lengthof the protein sequence, or even a fusion protein comprising two or moreepitopes from the HCV polyprotein. An epitope for use in the subjectinvention is not limited to a polypeptide having the exact sequence ofthe portion of the parent protein from which it is derived. Indeed,viral genomes are in a state of constant flux and contain severalvariable domains which exhibit relatively high degrees of variabilitybetween isolates. Thus the term “epitope” encompasses sequencesidentical to the native sequence, as well as modifications to the nativesequence, such as deletions, additions and substitutions (generallyconservative in nature).

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA81:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA82:178-182; Geysen et al. (1986) Molec. Immunol. 23:709-715, allincorporated herein by reference in their entireties. Using suchtechniques, a number of epitopes of HCV have been identified. See, e.g.,Chien et al., Viral Hepatitis and Liver Disease (1994) pp. 320-324, andfurther below. Similarly, conformational epitopes are readily identifiedby determining spatial conformation of amino acids such as by, e.g.,x-ray crystallography and 2-dimensional nuclear magnetic resonance. See,e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteinscan also be identified using standard antigenicity and hydropathy plots,such as those calculated using, e.g., the Omiga version 1.0 softwareprogram available from the Oxford Molecular Group. This computer programemploys the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA(1981) 78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots.

As used herein, the term “conformational epitope” refers to a portion ofa full-length protein, or an analog or mutein thereof, having structuralfeatures native to the amino acid sequence encoding the epitope withinthe full-length natural protein. Native structural features include, butare not limited to, glycosylation and three dimensional structure. Thelength of the epitope defining sequence can be subject to widevariations as these epitopes are believed to be formed by thethree-dimensional shape of the antigen (e.g., folding). Thus, aminoacids defining the epitope can be relatively few in number, but widelydispersed along the length of the molecule (or even on differentmolecules in the case of dimers, etc.), being brought into correctepitope conformation via folding. The portions of the antigen betweenthe residues defining the epitope may not be critical to theconformational structure of the epitope. For example, deletion orsubstitution of these intervening sequences may not affect theconformational epitope provided sequences critical to epitopeconformation are maintained (e.g., cysteines involved in disulfidebonding, glycosylation sites, etc.).

Conformational epitopes present in the NS3/4a region are readilyidentified using methods discussed above. Moreover, the presence orabsence of a conformational epitope in a given polypeptide can bereadily determined through screening the antigen of interest with anantibody (polyclonal serum or monoclonal to the conformational epitope)and comparing its reactivity to that of a denatured version of theantigen which retains only linear epitopes (if any). In such screeningusing polyclonal antibodies, it may be advantageous to absorb thepolyclonal serum first with the denatured antigen and see if it retainsantibodies to the antigen of interest. Additionally, in the case ofNS3/4a, a molecule which preserves the native conformation will alsohave protease and, optionally, helicase enzymatic activities. Suchactivities can be detected using enzymatic assays, as described furtherbelow.

Preferably, a conformational epitope is produced recombinantly and isexpressed in a cell from which it is extractable under conditions whichpreserve its desired structural features, e.g. without denaturation ofthe epitope. Such cells include bacteria, yeast, insect, and mammaliancells. Expression and isolation of recombinant conformational epitopesfrom the HCV polyprotein are described in e.g., InternationalPublication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734,which applications are herein incorporated by reference in theirentirety. Alternatively, it is possible to express the antigens andfurther renature the protein after recovery. It is also understood thatchemical synthesis may also provide conformational antigen mimitopesthat cross-react with the “native” antigen's conformational epitope.

The term “multiple epitope fusion antigen” or “MEFA” as used hereinintends a polypeptide in which multiple HCV antigens are part of asingle, continuous chain of amino acids, which chain does not occur innature. The HCV antigens may be connected directly to each other bypeptide bonds or may be separated by intervening amino acid sequences.The fusion antigens may also contain sequences exogenous to the HCVpolyprotein. Moreover, the HCV sequences present may be from multiplegenotypes and/or isolates of HCV. Examples of particular MEFAs for usein the present immunoassays are detailed in, e.g., InternationalPublication No. WO 97/44469, incorporated herein by reference in itsentirety, and are described further below.

An “antibody” intends a molecule that, through chemical or physicalmeans, specifically binds to a polypeptide of interest. Thus, an HCVcore antibody is a molecule that specifically binds to the HCV coreprotein. The term “antibody” as used herein includes antibodies obtainedfrom both polyclonal and monoclonal preparations, as well as, thefollowing: hybrid (chimeric) antibody molecules (see, for example,Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567);F(ab′)₂ and F(ab) fragments; Fv molecules (non-covalent heterodimers,see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096);single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988)Proc Natl Acad Sci USA 85:5879-5883); dimeric and trimeric antibodyfragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanizedantibody molecules (see, for example, Riechmann et al. (1988) Nature332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K.Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, anyfunctional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. Thus, the termencompasses antibodies obtained from murine hybridomas, as well as humanmonoclonal antibodies obtained using human rather than murinehybridomas. See, e.g., Cote, et al. Monclonal Antibodies and CancerTherapy, Alan R. Liss, 1985, p. 77.

A “recombinant” protein is a protein which retains the desired activityand which has been prepared by recombinant DNA techniques as describedherein. In general, the gene of interest is cloned and then expressed intransformed organisms, as described further below. The host organismexpresses the foreign gene to produce the protein under expressionconditions.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

By “equivalent antigenic determinant” is meant an antigenic determinantfrom different sub-species or strains of HCV, such as from strains 1, 2,or 3 of HCV. More specifically, epitopes are known, such as 5-1-1, andsuch epitopes vary between the strains 1, 2, and 3. Thus, the epitope5-1-1 from the three different strains are equivalent antigenicdeterminants and thus are “copies” even though their sequences are notidentical. In general the amino acid sequences of equivalent antigenicdeterminants will have a high degree of sequence homology, e.g., aminoacid sequence homology of more than 30%, preferably more than 40%, whenthe two sequences are aligned.

“Homology” refers to the percent similarity between two polynucleotideor two polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%-85%, preferably at least about 90%, and most preferably atleast about 95%-98% sequence similarity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100.

Readily available computer programs can be used to aid in the analysisof similarity and identity, such as ALIGN, Dayhoff, M. O. in Atlas ofProtein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358,National biomedical Research Foundation, Washington, D.C., which adaptsthe local homology algorithm of Smith and Waterman Advances in Appl.Math. 2:482-489, 1981 for peptide analysis. Programs for determiningnucleotide sequence similarity and identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent similarity of aparticular nucleotide sequence to a reference sequence can be determinedusing the homology algorithm of Smith and Waterman with a defaultscoring table and a gap penalty of six nucleotide positions.

Another method of establishing percent similarity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequencesimilarity.” Other suitable programs for calculating the percentidentity or similarity between sequences are generally known in the art,for example, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A transcription termination sequence may belocated 3′ to the coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their desiredfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper transcription factors, etc., are present. The promoter need notbe contiguous with the coding sequence, so long as it functions todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between thepromoter sequence and the coding sequence, as can transcribed introns,and the promoter sequence can still be considered “operably linked” tothe coding sequence.

A “control element” refers to a polynucleotide sequence which aids inthe expression of a coding sequence to which it is linked. The termincludes promoters, transcription termination sequences, upstreamregulatory domains, polyadenylation signals, untranslated regions,including 5′-UTRs and 3′-UTRs and when appropriate, leader sequences andenhancers, which collectively provide for the transcription andtranslation of a coding sequence in a host cell.

A “promoter” as used herein is a DNA regulatory region capable ofbinding RNA polymerase in a host cell and initiating transcription of adownstream (3′ direction) coding sequence operably linked thereto. Forpurposes of the present invention, a promoter sequence includes theminimum number of bases or elements necessary to initiate transcriptionof a gene of interest at levels detectable above background. Within thepromoter sequence is a transcription initiation site, as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eucaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes.

A control sequence “directs the transcription” of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

“Expression cassette” or “expression construct” refers to an assemblywhich is capable of directing the expression of the sequence(s) orgene(s) of interest. The expression cassette includes control elements,as described above, such as a promoter which is operably linked to (soas to direct transcription of) the sequence(s) or gene(s) of interest,and often includes a polyadenylation sequence as well. Within certainembodiments of the invention, the expression cassette described hereinmay be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude, one or more selectable markers, a signal which allows theplasmid construct to exist as single-stranded DNA (e.g., a M13 origin ofreplication), at least one multiple cloning site, and a “mammalian”origin of replication (e.g., a SV40 or adenovirus origin ofreplication).

“Transformation,” as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for insertion: for example, transformation by direct uptake,transfection, infection, and the like. For particular methods oftransfection, see further below. The exogenous polynucleotide may bemaintained as a nonintegrated vector, for example, an episome, oralternatively, may be integrated into the host genome.

A “host cell” is a cell which has been transformed, or is capable oftransformation, by an exogenous DNA sequence.

“Common solid support” intends a single solid matrix to which the HCVpolypeptides used in the subject immunoassays are bound covalently or bynoncovalent means such as hydrophobic adsorption.

“Immunologically reactive” means that the antigen in question will reactspecifically with anti-HCV antibodies present in a biological samplefrom an HCV-infected individual.

“Immune complex” intends the combination formed when an antibody bindsto an epitope on an antigen.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, including but not limited to, forexample, blood, plasma, serum, fecal matter, urine, bone marrow, bile,spinal fluid, lymph fluid, samples of the skin, external secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, organs, biopsies and also samples of in vitrocell culture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin,strepavidin or haptens) and the like. The term “fluorescer” refers to asubstance or a portion thereof which is capable of exhibitingfluorescence in the detectable range. Particular examples of labelswhich may be used under the invention include, but are not limited to,horse radish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl,umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol,NADPH and α-β-galactosidase.

II. Modes of Carrying out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of compositions and methods similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

As noted above, the present invention is based on the discovery of noveldiagnostic methods for accurately detecting early HCV infection. Themethods rely on the identification and use of highly immunogenic HCVantibodies and antigens which are present during the early stages of HCVseroconversion, thereby increasing detection accuracy and reducing theincidence of false results. The methods can be conveniently practiced ina single assay format.

More particularly, the assay is conducted on a solid support to whichhas been bound one or more HCV anti-core antibodies (directed againsteither the same or different HCV core epitopes) and an epitope derivedfrom the NS3/4a region of the HCV polyprotein. Examples of particularanti-core antibodies useful in the present invention include, but arenot limited to, antibody molecules such as monoclonal antibodies,directed against epitopes in the core region found between amino acids10-53; amino acids 10-45; amino acids 67-88; amino acids 120-130, orantibodies directed against any of the core epitopes identified in,e.g., Houghton et al., U.S. Pat. No. 5,350,671; Chien et al., Proc.Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent.Hepatol. (1993) 8:S33-39; Chien et al., International Publication No. WO93/00365; Chien, D. Y., International Publication No. WO 94/01778; andcommonly owned, allowed U.S. patent application Ser. Nos. 08/403,590 and08/444,818, the disclosures of which are incorporated herein byreference in their entireties.

The NS3/4a region of the HCV polyprotein has been described and theamino acid sequence and overall structure of the protein are disclosedin, e.g., Yao et al., Structure (November 1999) 7:1353-1363; Sali etal., Biochem. (1998) 37:3392-3401; and Bartenschlager, R., J. ViralHepat. (1999) 6:165-181. See, also, Dasmahapatra et al., U.S. Pat. No.5,843,752, incorporated herein by reference in its entirety. The subjectimmunoassays utilize at least one conformational epitope derived fromthe NS3/4a region that exists in the conformation as found in thenaturally occurring HCV particle or its infective product, as evidencedby the preservation of protease and, optionally, helicase enzymaticactivities normally displayed by the NS3/4a gene product and/orimmunoreactivity of the antigen with antibodies in a biological samplefrom an HCV-infected subject, and a loss of the epitope'simmunoreactivity upon denaturation of the antigen. For example, theconformational epitope can be disrupted by heating, changing the pH toextremely acid or basic, or by adding known organic denaturants, such asdithiothreitol (DTT) or an appropriate detergent. See, e.g., ProteinPurification Methods, a practical approach (E. L. V. Harris and S. Angaleds., IRL Press) and the denatured product compared to the product whichis not treated as above.

Protease and helicase activity may be determined using standard enzymeassays well known in the art. For example, protease activity may bedetermined using assays well known in the art. See, e.g., Takeshita etal., Anal. Biochem. (1997) 247:242-246; Kakiuchi et al., J. Biochem.(1997) 122:749-755; Sali et al., Biochemistry (1998) 37:3392-3401; Choet al., J. Virol. Meth. (1998) 72:109-115; Cerretani et al., Anal.Biochem. (1999) 266:192-197; Zhang et al., Anal. Biochem. (1999)270:268-275; Kakiuchi et al., J. Virol. Meth. (1999) 80:77-84; Fowler etal., J. Biomol. Screen. (2000) 5:153-158; and Kim et al., Anal. Biochem.(2000) 284:42-48. A particularly convenient assay for testing proteaseactivity is set forth in the examples below.

Similarly, helicase activity assays are well known in the art andhelicase activity of an NS3/4a epitope may be determined using, forexample, an ELISA assay, as described in, e.g., Hsu et al., Biochem.Biophys. Res. Commun. (1998) 253:594-599; a scintillation proximityassay system, as described in Kyono et al., Anal. Biochem. (1998)257:120-126; high throughput screening assays as described in, e.g.,Hicham et al., Antiviral Res. (2000) 46:181-193 and Kwong et al.,Methods Mol. Med. (2000) 24:97-116; as well as by other assay methodsknown in the art. See, e.g., Khu et al., J. Virol. (2001) 75:205-214;Utama et al., Virology (2000) 273:316-324; Paolini et al., J. Gen.Virol. (2000) 81:1335-1345; Preugschat et al., Biochemistry (2000)39:5174-5183; Preugschat et al., Methods Mol. Med. (1998) 19:353-364;and Hesson et al., Biochemistry (2000) 39:2619-2625.

The length of the antigen is sufficient to maintain an immunoreactiveconformational epitope. Often, the polypeptide containing the antigenused will be almost full-length, however, the polypeptide may also betruncated to, for example, increase solubility or to improve secretion.Generally, the conformational epitope found in NS3/4a is expressed as arecombinant polypeptide in a cell and this polypeptide provides theepitope in a desired form, as described in detail below.

Representative amino acid sequences for NS3/4a polypeptides are shown inFIG. 3 and FIGS. 4A through 4D. The bolded alanine occurring at position182 of FIG. 3 is substituted for the native serine found at thisposition in order to prevent autocatalyisis of the molecule that mightotherwise occur. The amino acid sequence shown at positions 2-686 ofFIGS. 4A through 4D corresponds to amino acid positions 1027-1711 ofHCV-1. An initiator codon (ATG) coding for Met, is shown as position 1.Additionally, the Thr normally occurring at position 1428 of HCV-1(amino acid position 403 of FIG. 4) is mutated to Pro, and the Sernormally occurring at position 1429 of HCV-1 (amino acid position 404 ofFIG. 4) is mutated to Ile. However, either the native sequence, with orwithout an N-terminal Met, the depicted analog, with or without theN-terminal Met, or other analogs and fragments can be used in thesubject assays, so long as the epitope is produced using a method thatretains or reinstates its native conformation such that proteaseactivity, and optionally, helicase activity is retained. Dasmahapatra etal., U.S. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. No. 5,990,276,both describe analogs of NS3/4a.

The NS3 protease of NS3/4a is found at about positions 1027-1207,numbered relative to HCV-1, positions 2-182 of FIG. 4. The structure ofthe NS3 protease and active site are known. See, e.g., De Francesco etal., Antivir. Ther. (1998) 3:99-109; Koch et al., Biochemistry (2001)40:631-640. Changes to the native sequence that will normally betolerated will be those outside of the active site of the molecule.Particularly, it is desirable to maintain amino acids 1- or 2-155 ofFIG. 4, with little or only conservative substitutions. Amino acidsoccurring beyond 155 will tolerate greater changes. Additionally, iffragments of the NS3/4a sequence found in FIG. 4 are used, thesefragments will generally include at least amino acids 1- or 2-155,preferably amino acids 1- or 2-175, and most preferably amino acids 1-or 2-182, with or without the N-terminal Met. The helicase domain isfound at about positions 1193-1657 of HCV-1 (positions 207-632 of FIG.4). Thus, if helicase activity is desired, this portion of the moleculewill be maintained with little or only conservative changes. One ofskill in the art can readily determine other regions that will toleratechange based on the known structure of NS3/4a.

The solid support may also comprise other antigens. For example,multiple epitope fusion antigens (termed “MEFAs”), as described inInternational Publication No. WO 97/44469, may be bound to the solidsupport for use in the subject assays. Such MEFAs include multipleepitopes derived from two or more of the various viral regions shown inFIG. 1 and Table 1. In particular, as shown in FIG. 1 and Table 1, AnHCV polyprotein, upon cleavage, produces at least ten distinct products,in the order of NH₂-Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Thecore polypeptide occurs at positions 1-191, numbered relative to HCV-1(see, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, forthe HCV-1 genome). This polypeptide is further processed to produce anHCV polypeptide with approximately amino acids 1-173. The envelopepolypeptides, E1 and E2, occur at about positions 192-383 and 384-746,respectively. The P7 domain is found at about positions 747-809. NS2 isan integral membrane protein with proteolytic activity and is found atabout positions 810-1026 of the polyprotein. NS2, either alone or incombination with NS3 (found at about positions 1027-1657), cleaves theNS2-NS3 sissle bond which in turn generates the NS3 N-terminus andreleases a large polyprotein that includes both serine protease and RNAhelicase activities. The NS3 protease, found at about positions1027-1207, serves to process the remaining polyprotein. The helicaseactivity is found at about positions 1193-1657. Completion ofpolyprotein maturation is initiated by autocatalytic cleavage at theNS3-NS4a junction, catalyzed by the NS3 serine protease. SubsequentNS3-mediated cleavages of the HCV polyprotein appear to involverecognition of polyprotein cleavage junctions by an NS3 molecule ofanother polypeptide. In these reactions, NS3 liberates an NS3 cofactor(NS4a, found about positions 1658-1711), two proteins (NS4b found atabout positions 1712-1972, and NS5a found at about positions 1973-2420),and an RNA-dependent RNA polymerase (NS5b found at about positions2421-3011).

TABLE 1 Domain Approximate Boundaries* C (core)  1-191 E1 192-383 E2384-746 P7 747-809 NS2  810-1026 NS3 1027-1657 NS4a 1658-1711 NS4b1712-1972 NS5a 1973-2420 NS5b 2421-3011 *Numbered relative to HCV-1.See, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2451-2455.

The multiple HCV antigens are part of a single, continuous chain ofamino acids, which chain does not occur in nature. Thus, the linearorder of the epitopes is different than their linear order in the genomein which they occur. The linear order of the sequences of the MEFAs foruse herein is preferably arranged for optimum antigenicity. Preferably,the epitopes are from more than one HCV strain, thus providing the addedability to detect multiple strains of HCV in a single assay. Thus, theMEFAs for use herein may comprise various immunogenic regions derivedfrom the polyprotein described above. Moreover, a protein resulting froma frameshift in the core region of the polyprotein, such as described inInternational Publication No. WO 99/63941, may be used in the MEFAs. Ifdesired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of one or moreepitopes derived from the HCV polyprotein may occur in the fusionprotein.

For example, epitopes derived from, e.g., the hypervariable region ofE2, such as a region spanning amino acids 384-410 or 390-410, can beincluded in the MEFA antigen. A particularly effective E2 epitope is onewhich includes a consensus sequence derived from this region, such asthe consensus sequenceGly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn(SEQ ID NO:6), which represents a consensus sequence for amino acids390-410 of the HCV type 1 genome. A representative E2 epitope present ina MEFA of the invention can comprise a hybrid epitope spanning aminoacids 390-444. Such a hybrid E2 epitope can include a consensus sequencerepresenting amino acids 390-410 fused to the native amino acid sequencefor amino acids 411-444 of HCV E2.

Additionally, the antigens may be derived from various HCV strains.Multiple viral strains of HCV are known, and epitopes derived from anyof these strains can be used in a fusion protein. It is well known thatany given species of organism varies from one individual organism toanother and further that a given organism such as a virus can have anumber of different strains. For example, as explained above, HCVincludes at least 6 genotypes. Each of these genotypes includesequivalent antigenic determinants. More specifically, each strainincludes a number of antigenic determinants that are present on allstrains of the virus but are slightly different from one viral strain toanother. For example, HCV includes the antigenic determinant known as5-1-1 (See, FIG. 1). This particular antigenic determinant appears inthree different forms on the three different viral strains of HCV.Accordingly, in a preferred embodiment of the invention all three formsof 5-1-1 appear on the multiple epitope fusion antigen used in thesubject immunoassays. Similarly, equivalent antigenic determinants fromthe core region of different HCV strains may also be present. Ingeneral, equivalent antigenic determinants have a high degree ofhomology in terms of amino acid sequence which degree of homology isgenerally 30% or more, preferably 40% or more, when aligned. Themultiple copy epitope of the present invention can also include multiplecopies which are exact copies of the same epitope.

Representative MEFAs for use with the present assays are described inInternational Publication No. WO 97/44469. Additional representativeMEFAs for use herein include those termed MEFA 12, MEFA 13 and MEFA13.1. It is to be understood that these MEFAs are merely representativeand other epitopes derived from the HCV genome will also find use withthe present assays and may be incorporated into these or other MEFAs.

The DNA sequence and corresponding amino acid sequence of MEFA 12 isshown in FIGS. 7A through 7F. The general structural formula for MEFA 12is shown in FIG. 6 and is as follows: hSOD-E1(type 1)-E2 HVR consensus(type 1a)-E2 HVR consensus (types 1 and 2)-c33c short (type1)-5-1-1(type 1)-5-1-1(type 3)-5-1-1(type 2)-c100(type 1)-NS5(type1)-NS5(type 1)-core (types 1+2)-core (types 1+2). This multiple copyepitope includes the following amino acid sequence, numbered relative toHCV-1 (the numbering of the amino acids set forth below follows thenumbering designation provided in Choo, et al. (1991) Proc. Natl. Acad.Sci. USA 88:2451-2455, in which amino acid #1 is the first methionineencoded by the coding sequence of the core region): amino acids 1-69 ofsuperoxide dismutase (SOD, used to enhance recombinant expression of theprotein); amino acids 303 to 320 of the polyprotein from the E1 region;amino acids 390 to 410 of the polyprotein, representing a consensussequence for the hypervariable region of HCV-1a E2; amino acids 384 to414 of the polyprotein from region E2, representing a consensus sequencefor the E2 hypervariable regions of HCV-1 and HCV-2; amino acids¹²¹I-1457 of the HCV-1 polyprotein which define the helicase; threecopies of an epitope from 5-1-1, amino acids 1689-1735, one from HCV-1,one from HCV-3 and one from HCV-2, which copies are equivalent antigenicdeterminants from the three different viral strains of HCV; HCVpolypeptide C100 of HCV-1, amino acids 1901-1936 of the polyprotein; twoexact copies of an epitope from the NS5 region of HCV-1, each with aminoacids 2278 to 2313 of the HCV polyprotein; and two copies of threeepitopes from the core region, two from HCV-1 and one from HCV-2, whichcopies are equivalent antigenic determinants represented by amino acids9 to 53 and 64-88 of HCV-1 and 67-84 of HCV-2.

Table 2 shows the amino acid positions of the various epitopes in MEFA12 with reference to FIGS. 7A through 7F herein. The numbering in thetables is relative to HCV-1. See, Choo et al. (1991) Proc. Natl. Acad.Sci. USA 88:2451-2455. MEFAs 13 and 13.1 also share the general formulaspecified above for MEFA 12, with modifications as indicated in Tables 3and 4, respectively.

TABLE 2 MEFA 12 mefa aa# 5′ end site epitope hcv aa# strain   1-69* Nco1hSOD 72-89 MluI E1 303-320 1  92-112 Hind111 E2 HVR1a 390-410 1consensus 113-143 E2 HVR1 + 2 384-414 1, 2 consensus 146-392 SpeI C33Cshort 1211-1457 1 395-441 SphI 5-1-1 1689-1735 1 444-490 NruI 5-1-11689-1735 3 493-539 ClaI 5-1-1 1689-1735 2 542-577 AvaI C100 1901-1936 1580-615 XbaI NS5 2278-2313 1 618-653 BglII NS5 2278-2313 1 654-741 NcoIcore 9-53, R47L 1 epitopes 64-88 1 67-84 2 742-829 BalI core 9-53, R47L1 epitopes 64-88 1 67-84 2 *The SOD protein is truncated so that so thatthe detection conjugate, an HRP-labeled anti-SOD antibody does not bindthe MEFA. The core epitope is mutated to prevent the antibodies to HCVcore, used in detection, from binding to the MEFA.

TABLE 3 MEFA 13 mefa aa# 5′ end site epitope hcv aa# strain  1-156 Nco1mutated hSOD (aa 70-72, ALA) 161-178 MluI E1 303-320 1 181-201 Hind111E2 HVR1a 390-410 1 consensus 202-232 E2 HVR1 + 2 384-414 1, 2 consensus235-451 C33C short 1211-1457 1 454-500 HindIII 5-1-1 PImut* 1689-1735 1503-549 NruI 5-1-1 PImut* 1689-1735 3 552-598 ClaI 5-1-1 PImut*1689-1735 2 601-636 AvaI C100 1901-1936 1 639-674 XbaI NS5 2278-2313 1677-712 BglII NS5 2278-2313 1 713-800 core 9-53, R47L 1 epitopes 64-88 167-84 2 801-888 core 9-53, R47L 1 epitopes 64-88 1 67-84 2 *The 5-1-1epitopes are modified by eliminating possible cleavage sites (CS or CA)targeted by the NS3/4a recombinant protein. Instead of CS or CA, thesequence has been changed to PI. Additionally, the SOD protein ismutated so that the detection conjugate, an HRP-labeled anti-SODantibody does not bind the MEFA. The core epitope is mutated to preventthe antibodies to HCV core, used in detection, from binding to the MEFA.

TABLE 4 MEFA 13.1 mefa aa# 5′ end site epitope hcv aa# strain  1-86 NcoImutated hSOD (aa 70-72, ALA)  89-106 MluI E1 303-320 1 109-129 HindIIIE2 HVR1a 390-410 1 consensus 130-160 E2 HVR1 + 2 384-414 1, 2 consensus163-379 C33C short 1211-1457 1 382-428 HindIII 5-1-1 PImut* 1689-1735 1431-477 NruI 5-1-1 PImut* 1689-1735 3 480-526 ClaI 5-1-1 PImut*1689-1735 2 529-564 AvaI C100 1901-1936 1 567-602 XbaI NS5 2278-2313 1605-640 BglII NS5 2278-2313 1 641-728 core 9-53, R47L 1 epitopes 64-88 167-84 2 729-816 core 9-53, R47L 1 epitopes 64-88 1 67-84 2 *The 5-1-1epitopes are modified by eliminating possible cleavage sites (CS or CA)targeted by the NS3/4a recombinant protein. Instead of CS or CA, thesequence has been changed to PI. Additionally, the SOD protein ismutated so that the detection conjugate, an HRP-labeled anti-SODantibody does not bind the MEFA. The core epitope is mutated to preventthe antibodies to HCV core, used in detection, from binding to the MEFA.

In one assay format, the sample is combined with the solid support, asdescribed further below. If the sample is infected with HCV, coreantigens, as well as HCV antibodies to those epitopes present on thesolid support, will bind to the solid support components. A detectablylabeled anti-core antibody is then added. The labeled anti-core antibodyis directed against a different epitope than the anti-core antibody thatis bound to the solid support. This anti-core antibody binds the coreantigen captured by the anti-core antibodies on the solid support.

An antigen that reacts with the captured HCV antibody from thebiological sample, which captured sample HCV antibody is reactive withthe NS3/4a epitope, is also added. This antigen is preferably an epitopederived from the NS3 region of the HCV polyprotein. This antigen bindsthe captured HCV antibody from the sample. A number of antigensincluding such epitopes are known, including, but not limited toantigens derived from the c33c and c100 regions, as well as fusionproteins comprising an NS3 epitope, such as c25. These and other NS3epitopes are useful in the present assays and are known in the art anddescribed in, e.g., Houghton et al, U.S. Pat. No. 5,350,671; Chien etal., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J.Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., InternationalPublication No. WO 93/00365; Chien, D. Y., International Publication No.WO 94/01778; and commonly owned, allowed U.S. patent application Ser.Nos. 08/403,590 and 08/444,818, the disclosures of which areincorporated herein by reference in their entireties.

A second labeled antibody, directed against the antigen described above,is added. This antibody can be directed against any epitope included inthe antigen. For example, the antibody can be directed against the NS3region present in the antigen. Alternatively, if the antigen above isexpressed as a fusion protein, the second labeled antibody can bedirected against the fusion partner. Additional antigens and antibodiescan be added to the assay, particularly if the solid support includes aMEFA. These assay formats are explained further below.

A representative assay under the invention is depicted in FIG. 2. Asshown in the figure, the solid support includes two anti-core monoclonalantibodies, termed c11-3 and c11-7. These antibodies are directedagainst an epitope found in the N-terminal region of the core protein atamino acids 10-53, numbered relative to the HCV1 polyprotein sequence.The solid support also includes an epitope to NS3/4a. The biologicalsample is added to the solid support. HCV core antigen, as well asantibodies directed against the NS3/4a epitope, both present in thesample, will bind the capture reagents on the solid support.

Horse radish peroxidase (HRP)-labeled anti-core monoclonal antibodyc11-14, directed against a C-terminal region of the core found at aminoacid positions 120-130, numbered relative to the HCV1 polyproteinsequence, is then added. A fusion protein, comprising a sequence fromhuman SOD (hSOD) and an epitope from the c33c region is added, as is asecond HRP-labeled antibody, directed against the SOD portion of thefusion protein. The SOD-c33c fusion will bind to the anti-NS3 antibodyand the anti-SOD antibody will, in turn, bind the SOD-c33c fusionprotein. Detection of the label indicates the presence of HCV infection.

Another representative assay under the invention is depicted in FIG. 8.The antibody assay configuration is an antigen-antibody-antigen sandwichcapture assay using both NS3/4a and MEFA 12. The solid support includesthe two anti-core monoclonal antibodies described above, an epitope toNS3/4a, as well as a representative MEFA, MEFA 12, which includes atruncated version of human SOD. As with the assay above, the biologicalsample is added to the solid support. HCV core antigen, as well asantibodies directed against the NS3/4a epitope and epitopes of the MEFA,present in the sample, will bind the capture reagents on the solidsupport. Two antigens, one reactive with sample antibodies that bindNS3/4a (as described above) and one reactive with sample antibodies thatbind MEFA 12, are added. In FIG. 8, the antigen reactive with the MEFA12/sample antibody complex, is a fusion between an SOD molecule andc22ks Δ47-L44W. The c22ks antigen is from the core region and includesamino acids Lys₁₀ to Ser₉₉ of the polyprotein, as well as a deletion ofArg47 normally present and a substitution of Leu for Trp at position 44.The antibody detection conjugate is the second HRP-labeled monoclonalanti-SOD antibody, described above.

The above-described antigen/antibody combination assays are particularlyadvantageous as both the HCV core antigen and antibodies to NS3/4aand/or core may be detected by the same support in the same assay.Moreover, as described above, additional HCV epitopes, such as SOD-fusedto c100, 5-1-1, NS5 antigens, as well as a protein resulting from aframeshift in the core region of the polyprotein, such as described inInternational Publication No. WO 99/63941, may be used in thecombination cocktail to cover other non-structural epitopes of HCV.

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding production of antibodies for usein the subject immunoassays; production of polypeptides for use in theimmunoassays; and methods of conducting the immunoassays.

Production of Antibodies for Use in the HCV Immunoassays

As explained above, the assay utilizes various antibodies which arebound to a solid support (e.g., one or more anti-core antibodies), andthat detect antigen/antibody complexes formed when HCV infection ispresent in the sample. These antibodies may be polyclonal or monoclonalantibody preparations, monospecific antisera, human antibodies, or maybe hybrid or chimeric antibodies, such as humanized antibodies, alteredantibodies, F(ab′)₂ fragments, F(ab) fragments, Fv fragments,single-domain antibodies, dimeric or trimeric antibody fragmentconstructs, minibodies, or functional fragments thereof which bind tothe antigen in question.

Antibodies are produced using techniques well known to those of skill inthe art and disclosed in, for example, U.S. Pat. Nos. 4,011,308;4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745. For example,polyclonal antibodies are generated by immunizing a suitable animal,such as a mouse, rat, rabbit, sheep or goat, with an antigen ofinterest. In order to enhance immunogenicity, the antigen can be linkedto a carrier prior to immunization. Such carriers are well known tothose of ordinary skill in the art. Immunization is generally performedby mixing or emulsifying the antigen in saline, preferably in anadjuvant such as Freund's complete adjuvant, and injecting the mixtureor emulsion parenterally (generally subcutaneously or intramuscularly).The animal is generally boosted 2-6 weeks later with one or moreinjections of the antigen in saline, preferably using Freund'sincomplete adjuvant. Antibodies may also be generated by in vitroimmunization, using methods known in the art. Polyclonal antiserum isthen obtained from the immunized animal. See, e.g., Houghton et al.,U.S. Pat. No. 5,350,671, for a description of the production of anti-HCVpolyclonal antibodies.

Monoclonal antibodies are generally prepared using the method of Kohlerand Milstein (1975) Nature 256:495-497, or a modification thereof.Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of nonspecifically adherent cells) by applying a cell suspensionto a plate or well coated with the antigen. B-cells, expressingmembrane-bound immunoglobulin specific for the antigen, will bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,“HAT”). The resulting hybridomas are plated by limiting dilution, andare assayed for the production of antibodies which bind specifically tothe immunizing antigen (and which do not bind to unrelated antigens).The selected monoclonal antibody-secreting hybridomas are then culturedeither in vitro (e.g., in tissue culture bottles or hollow fiberreactors), or in vivo (e.g., as ascites in mice).

The production of various anti-HCV monoclonal antibodies has beendescribed in, e.g., Houghton et al., U.S. Pat. No. 5,350,671; Chien etal., International Publication No. WO 93/00365; commonly owned, allowedU.S. patent application Ser. Nos. 08/403,590 and 08/444,818; andKashiwakuma et al., U.S. Pat. No. 5,871,904, incorporated herein byreference in their entireties.

As explained above, antibody fragments which retain the ability torecognize the antigen of interest, will also find use in the subjectimmunoassays. A number of antibody fragments are known in the art whichcomprise antigen-binding sites capable of exhibiting immunologicalbinding properties of an intact antibody molecule. For example,functional antibody fragments can be produced by cleaving a constantregion, not responsible for antigen binding, from the antibody molecule,using e.g., pepsin, to produce F(ab′)₂ fragments. These fragments willcontain two antigen binding sites, but lack a portion of the constantregion from each of the heavy chains. Similarly, if desired, Fabfragments, comprising a single antigen binding site, can be produced,e.g., by digestion of polyclonal or monoclonal antibodies with papain.Functional fragments, including only the variable regions of the heavyand light chains, can also be produced, using standard techniques suchas recombinant production or preferential proteolytic cleavage ofimmunoglobulin molecules. These fragments are known as F_(v). See, e.g.,Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman etal. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem19:4091-4096.

A single-chain Fv (“sFv” or “scFv”) polypeptide is a covalently linkedV_(H)-V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883. A number ofmethods have been described to discern and develop chemical structures(linkers) for converting the naturally aggregated, but chemicallyseparated, light and heavy polypeptide chains from an antibody V regioninto an sFv molecule which will fold into a three dimensional structuresubstantially similar to the structure of an antigen-binding site. See,e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. The sFvmolecules may be produced using methods described in the art. See, e.g.,Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883; U.S. Pat.Nos. 5,091,513, 5,132,405 and 4,946,778. Design criteria includedetermining the appropriate length to span the distance between theC-terminus of one chain and the N-terminus of the other, wherein thelinker is generally formed from small hydrophilic amino acid residuesthat do not tend to coil or form secondary structures. Such methods havebeen described in the art. See, e.g., U.S. Pat. Nos. 5,091,513,5,132,405 and 4,946,778. Suitable linkers generally comprise polypeptidechains of alternating sets of glycine and serine residues, and mayinclude glutamic acid and lysine residues inserted to enhancesolubility.

“Mini-antibodies” or “minibodies” will also find use with the presentinvention. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region. Pack et al. (1992) Biochem 31:1579-1584. Theoligomerization domain comprises self-associating α-helices, e.g.,leucine zippers, that can be further stabilized by additional disulfidebonds. The oligomerization domain is designed to be compatible withvectorial folding across a membrane, a process thought to facilitate invivo folding of the polypeptide into a functional binding protein.Generally, minibodies are produced using recombinant methods well knownin the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B: 120-126.

Production of Antigens for Use in the HCV Immunoassays

As explained above, the molecules of the present invention are generallyproduced recombinantly. Thus, polynucleotides encoding HCV antigens foruse with the present invention can be made using standard techniques ofmolecular biology. For example, polynucleotide sequences coding for theabove-described molecules can be obtained using recombinant methods,such as by screening cDNA and genomic libraries from cells expressingthe gene, or by deriving the gene from a vector known to include thesame. Furthermore, the desired gene can be isolated directly from viralnucleic acid molecules, using techniques described in the art, such asin Houghton et al., U.S. Pat. No. 5,350,671. The gene of interest canalso be produced synthetically, rather than cloned. The molecules can bedesigned with appropriate codons for the particular sequence. Thecomplete sequence is then assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984)Science 223:1299; and Jay et al. (1984) J. Biol. Chem. 259:6311.

Thus, particular nucleotide sequences can be obtained from vectorsharboring the desired sequences or synthesized completely or in partusing various oligonucleotide synthesis techniques known in the art,such as site-directed mutagenesis and polymerase chain reaction (PCR)techniques where appropriate. See, e.g., Sambrook, supra. In particular,one method of obtaining nucleotide sequences encoding the desiredsequences is by annealing complementary sets of overlapping syntheticoligonucleotides produced in a conventional, automated polynucleotidesynthesizer, followed by ligation with an appropriate DNA ligase andamplification of the ligated nucleotide sequence via PVR. See, e.g.,Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4084-4088.Additionally, oligonucleotide directed synthesis (Jones et al. (1986)Nature 54:75-82), oligonucleotide directed mutagenesis of pre-existingnucleotide regions (Riechmann et al. (1988) Nature 332:323-327 andVerhoeyen et al. (1988) Science 239:1534-1536), and enzymatic filling-inof gapped oligonucleotides using T₄ DNA polymerase (Queen et al. (1989)Proc. Natl. Acad. Sci. USA 86:10029-10033) can be used under theinvention to provide molecules having altered or enhancedantigen-binding capabilities, and/or reduced immunogenicity.

Once coding sequences have been prepared or isolated, such sequences canbe cloned into any suitable vector or replicon. Numerous cloning vectorsare known to those of skill in the art, and the selection of anappropriate cloning vector is a matter of choice. Suitable vectorsinclude, but are not limited to, plasmids, phages, transposons, cosmids,chromosomes or viruses which are capable of replication when associatedwith the proper control elements.

The coding sequence is then placed under the control of suitable controlelements, depending on the system to be used for expression. Thus, thecoding sequence can be placed under the control of a promoter, ribosomebinding site (for bacterial expression) and, optionally, an operator, sothat the DNA sequence of interest is transcribed into RNA by a suitabletransformant. The coding sequence may or may not contain a signalpeptide or leader sequence which can later be removed by the host inpost-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;4,425,437; 4,338,397.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the sequencesrelative to the growth of the host cell. Regulatory sequences are knownto those of skill in the art, and examples include those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of a regulatory compound.Other types of regulatory elements may also be present in the vector.For example, enhancer elements may be used herein to increase expressionlevels of the constructs. Examples include the SV40 early gene enhancer(Dijkema et al. (1985) EMBO J. 4:761), the enhancer/promoter derivedfrom the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman etal. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and elements derived fromhuman CMV (Boshart et al. (1985) Cell 41:521), such as elements includedin the CMV intron A sequence (U.S. Pat. No. 5,688,688). The expressioncassette may further include an origin of replication for autonomousreplication in a suitable host cell, one or more selectable markers, oneor more restriction sites, a potential for high copy number and a strongpromoter.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the “control” of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the sequences encodingthe molecule of interest may be desirable to achieve this end. Forexample, in some cases it may be necessary to modify the sequence sothat it can be attached to the control sequences in the appropriateorientation; i.e., to maintain the reading frame. The control sequencesand other regulatory sequences may be ligated to the coding sequenceprior to insertion into a vector. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

As explained above, it may also be desirable to produce mutants oranalogs of the antigen of interest. This is particularly true withNS3/4a. Methods for doing so are described in, e.g., Dasmahapatra etal., U.S. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. No. 5,990,276.Mutants or analogs of this and other HCV proteins for use in the subjectassays may be prepared by the deletion of a portion of the sequenceencoding the polypeptide of interest, by insertion of a sequence, and/orby substitution of one or more nucleotides within the sequence.Techniques for modifying nucleotide sequences, such as site-directedmutagenesis, and the like, are well known to those skilled in the art.See, e.g., Sambrook et al., supra; Kunkel, T. A. (1985) Proc. Natl.Acad. Sci. USA (1985) 82:448; Geisselsoder et al. (1987) BioTechniques5:786; Zoller and Smith (1983) Methods Enzymol. 100:468;Dalbie-McFarland et al. (1982) Proc. Natl. Acad. Sci USA 79:6409.

The molecules can be expressed in a wide variety of systems, includinginsect, mammalian, bacterial, viral and yeast expression systems, allwell known in the art.

For example, insect cell expression systems, such as baculovirussystems, are known to those of skill in the art and described in, e.g.,Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, interalia, Invitrogen, San Diego Calif. (“MaxBac” kit). Similarly, bacterialand mammalian cell expression systems are well known in the art anddescribed in, e.g., Sambrook et al., supra. Yeast expression systems arealso known in the art and described in, e.g., Yeast Genetic Engineering(Barr et al., eds., 1989) Butterworths, London.

A number of appropriate host cells for use with the above systems arealso known. For example, mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human embryonic kidney cells, human hepatocellularcarcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”)cells, as well as others. Similarly, bacterial hosts such as E. coli,Bacillus subtilis, and Streptococcus spp., will find use with thepresent expression constructs. Yeast hosts useful in the presentinvention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Nucleic acid molecules comprising nucleotide sequences of interest canbe stably integrated into a host cell genome or maintained on a stableepisomal element in a suitable host cell using various gene deliverytechniques well known in the art. See, e.g., U.S. Pat. No. 5,399,346.

Depending on the expression system and host selected, the molecules areproduced by growing host cells transformed by an expression vectordescribed above under conditions whereby the protein is expressed. Theexpressed protein is then isolated from the host cells and purified. Ifthe expression system secretes the protein into growth media, theproduct can be purified directly from the media. If it is not secreted,it can be isolated from cell lysates. The selection of the appropriategrowth conditions and recovery methods are within the skill of the art.

The recombinant production of various HCV antigens has been described.See, e.g., Houghton et al., U.S. Pat. No. 5,350,671; Chien et al., J.Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., InternationalPublication No. WO 93/00365; Chien, D. Y., International Publication No.WO 94/01778.

Immunodiagnostic Assays

Once produced, the above anti-core antibodies and NS3/4a antigens areplaced on an appropriate solid support for use in the subjectimmunoassays. A solid support, for the purposes of this invention, canbe any material that is an insoluble matrix and can have a rigid orsemi-rigid surface. Exemplary solid supports include, but are notlimited to, substrates such as nitrocellulose (e.g., in membrane ormicrotiter well form); polyvinylchloride (e.g., sheets or microtiterwells); polystyrene latex (e.g., beads or microtiter plates);polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, magnetically responsive beads, and the like. Particular supportsinclude plates, pellets, disks, capillaries, hollow fibers, needles,pins, solid fibers, cellulose beads, pore-glass beads, silica gels,polystyrene beads optionally cross-linked with divinylbenzene, graftedco-poly beads, polyacrylamide beads, latex beads, dimethylacrylamidebeads optionally crosslinked with N-N′-bis-acryloylethylenediamine, andglass particles coated with a hydrophobic polymer.

If desired, the molecules to be added to the solid support can readilybe functionalized to create styrene or acrylate moieties, thus enablingthe incorporation of the molecules into polystyrene, polyacrylate orother polymers such as polyimide, polyacrylamide, polyethylene,polyvinyl, polydiacetylene, polyphenylene-vinylene, polypeptide,polysaccharide, polysulfone, polypyrrole, polyimidazole, polythiophene,polyether, epoxies, silica glass, silica gel, siloxane, polyphosphate,hydrogel, agarose, cellulose, and the like.

In one context, a solid support is first reacted with the HCV anti-coreantibodies and NS3/4a epitope (collectively called “the solid-phasecomponents” herein), and optionally, one or more MEFAs, under suitablebinding conditions such that the molecules are sufficiently immobilizedto the support. Sometimes, immobilization to the support can be enhancedby first coupling the antigen and/or antibody to a protein with bettersolid phase-binding properties. Suitable coupling proteins include, butare not limited to, macromolecules such as serum albumins includingbovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulinmolecules, thyroglobulin, ovalbumin, and other proteins well known tothose skilled in the art. Other reagents that can be used to bindmolecules to the support include polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andthe like. Such molecules and methods of coupling these molecules toantigens, are well known to those of ordinary skill in the art. See,e.g., Brinkley, M. A. (1992) Bioconjugate Chem. 3:2-13; Hashida et al.(1984) J. Appl. Biochem. 6:56-63; and Anjaneyulu and Staros (1987)International J. of Peptide and Protein Res. 30:117-124.

After reacting the solid support with the solid-phase components, anynonimmobilized solid-phase components are removed from the support bywashing, and the support-bound components are then contacted with abiological sample suspected of containing HCV antibodies and antigens(collectively called “ligand molecules” herein) under suitable bindingconditions. After washing to remove any nonbound ligand molecules, asecond anti-core antibody, directed against a different epitope than theanti-core antibody bound to the support, is added under suitable bindingconditions. The added anti-core antibody includes a detectable label, asdescribed above, and acts to bind any core antigen that might be presentin the sample which has reacted with the support-bound anti-coreantibody. Also added are one or more antigens that can react withantibodies present in the sample that have, in turn, reacted with theNS3/4A epitope. As explained above, the antigen is typically derivedfrom the NS3 region of the HCV polyprotein, and particularly from thec33c region of HCV. See, Houghton et al, U.S. Pat. No. 5,350,671; Chienet al., Proc. Natl. Acad. Sci. (1989) 89:10011-10015; InternationalPublication No. WO 93/00365; and commonly owned, allowed U.S. patentapplication Ser. Nos. 08/403,590 and 08/444,818, for a description ofthis region and epitopes derived therefrom. A labeled antibody directedagainst this antigen is also added. The antibody will therefore bind theantigen, which has reacted with anti-NS3 antibodies present in thesample. For this purpose, the c33c epitope can be conveniently providedas a fusion between c33c and human superoxide dismutase (hSOD), producedrecombinantly e.g., by methods described in Houghton et al., U.S. Pat.No. 5,350,671. The nucleotide and amino acid sequences for human SOD areknown and reported in Hallewell et al., U.S. Pat. No. 5,710,033. Alabeled antibody directed against human SOD can therefore be used todetect the presence of complexes formed between the NS3/4a epitope, anyantibodies in the sample which react with this epitope, and HCVpolypeptides which in turn bind the antibody in the sample.

If a MEFA is present on the solid support, one or more additionalantigens, reactive with antibodies from the biological sample which arebound to antigens present on the MEFA, may also be added to the assay.Particularly useful in this context is an antigen derived from the coreregion of HCV, and more particularly, from the c22 antigen whichincludes 119 N-terminal core amino acids of the HCV polyprotein. Oneparticular antigen derived from c22 is c22ks Δ47-L44W which includesamino acids Lys₁₀ to Ser₉₉ of the polyprotein, as well as a deletion ofArg47 normally present and a substitution of Leu for Trp at position 44.As with the c33c epitope described above, this antigen can be providedas a fusion with hSOD and the same labeled antibody, directed againsthuman SOD, can be used to detect the presence of complexes formedbetween antibodies present in the sample and the NS3/4a epitope and/orthe MEFA, which complexes are also bound with the HCV antigens (e.g.,c33c and c22).

More particularly, an ELISA method can be used, wherein the wells of amicrotiter plate are coated with the solid-phase components. Abiological sample containing or suspected of containing ligand moleculesis then added to the coated wells. After a period of incubationsufficient to allow ligand-molecule binding to the immobilizedsolid-phase component, the plate(s) can be washed to remove unboundmoieties and a detectably labeled secondary binding molecule (labeledanti-core antibody), an NS3 epitope-containing molecule, and an antibodydirected against the NS3 epitope-containing molecule added. Thesemolecules are allowed to react with any captured sample antigen andantibody, the plate washed and the presence of the labeled antibodiesdetected using methods well known in the art.

The above-described assay reagents, including the immunoassay solidsupport with bound antibodies and antigens, as well as antibodies andantigens to be reacted with the captured sample, can be provided inkits, with suitable instructions and other necessary reagents, in orderto conduct immunoassays as described above. The kit can also contain,depending on the particular immunoassay used, suitable labels and otherpackaged reagents and materials (i.e. wash buffers and the like).Standard immunoassays, such as those described above, can be conductedusing these kits.

III. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 HCV Antigen/Antibody Combination Immunoassay

The present HCV antigen/antibody combination immunoassay was compared toother HCV assays to test the seroconversion detection limits and comparethese limits to those obtained in other commercially available assays asfollows.

A. Materials and Methods

Blood Samples: Panels of commercially available human blood samples wereused. Such panels are available from, e.g., Boston Biomedica, Inc., WestBridgewater, Mass. (BBI); Bioclinical Partners, Franklin, Mass. (BCP);and North American Biologics, Inc., BocoRatan, Fla. (NABI). The daysindicated in Tables 5 and 6 are days on which blood was collected fromthe subjects.

Monoclonal Antibodies: Monoclonal antibodies c11-3, c11-7 and c 11-14were obtained from Ortho Clinical Diagnostics, Raritan, N.J. The c11-3and c11-7 antibodies are directed against an N-terminal portion of thecore (amino acids 10-53, numbered relative to the HCV1 polyprotein).Monoclonal antibody c11-14 is directed against a C-terminal portion ofthe core (amino acids 120-130, numbered relative to the HCV1polyprotein). The c11-14 antibody was conjugated to horse radishperoxidase (HRP) using standard procedures.

Monoclonal antibody 5A-3 is an anti-SOD antibody directed against aminoacids 1 to 65 of SOD and was made using standard techniques. Theantibody was conjugated to HRP as described above.

B. Antigens:

The c33c antigen (266 amino acids, amino acids 1192 to 1457 of the HCV1polyprotein) was expressed as an internal SOD fusion polypeptide in E.coli by methods described for the synthesis of the 5-1-1 antigen (Choo,et al., Science (1989) 244:359-362, herein incorporated by reference).The recombinant antigen was purified as described in Chien, et al.,Proc. Natl. Acad. Sci. (1989) 89:10011-10015 (herein incorporated byreference). See, also, Houghton et al., U.S. Pat. No. 5,350,671, forproduction protocols for SOD-c33c.

The NS3/4a epitope used in the assay is a conformational epitope havingthe sequence specified in FIG. 3.

C. Immunoassay Formats:

The Abbott PRISM assay (Abbott Laboratories, Abbott Park, Ill.), iscommercially available and is an antibody-based detection assay. Theassay was performed using the manufacturer's instructions.

The ORTHO HCV Version 3.0 ELISA Test System (termed Ortho 3.0 assayherein, Ortho Clinical Diagnostics, Raritan, N.J.) is an antibody-baseddetection assay. The assay was conducted using the manufacturer'sinstructions.

The Roche Amplicor assay (Roche, Pleasant, Calif.) is a commerciallyavailable PCR-based assay. The assay was performed using themanufacturer's instructions.

The Gen-Probe TMA assay (San Diego, Calif.) is a commercially availabletranscription-mediated amplification assay. The assay was performedusing the manufacturer's instructions.

The Ortho antigen assay (Ortho Clinical Diagnostics, Raritan, N.J.) isan antigen-based detection assay. The assay was performed using themanufacturer's instructions.

The subject HCV antigen/antibody combination immunoassay was performedas follows. 4 mg/mL each of purified monoclonal antibodies C11-7 andC11-3 in 1× phosphate-buffered saline (PBS), pH 7.4 were combined andmixed well. 90 ng of the NS3/4a recombinant antigen was added to thesame coating buffer. The solution was mixed for 30 minutes prior tocoating. 200 mL of the above solution was added per well to 96-wellCostar medium binding microtiter plates (Corning, Inc.) Plates wereincubated at 15-30° C. for 16-24 hours. Plates were washed two timeswith dH₂O, followed with 300 μL/well postcoat buffer (1% bovine serumalbumin (BSA), 1×PBS) for 1 hour and 300 μl/well stability buffer(1×PBS, 1% BSA, mannitol, polyethylene glycol (PEG), gelatin) for 1hour. Plates were aspirated and dried at 4° C. in a lyophilizer for 24hours. Plates were pouched with desiccant.

To conduct the antigen/antibody combination immunoassay, 100 μL ofenhanced lysis buffer (1% N-laurylsarcosine, 0.65M NaCl, 50 mg/mL mouseIgG technical grade (Sigma, St. Louis, Mo.), 1% BSA sulfhydryl-modified(Bayer), 0.1% Casein) were added to the plate. 100 mL of sample werethen added. This was incubated on a shaker at 40° C. for one hour. Theplates were washed six times with 1×PBS, 0.1% Tween-20, on an OrthoPlate Washer. 200 mL conjugate solution (1:75 dilution c11-14-HRP with250 ng/assay SOD-c33c antigen plus 1:5000 dilution mouse anti-SOD-HRP inHCV 3.0 sample diluent (from ORTHO HCV Version 3.0 ELISA Test System,Ortho Clinical Diagnostics, Raritan, N.J.) without SOD extract, allprepared 30 minutes prior to addition). The solution was incubated 45minutes with shaking at 40° C. This was washed six times, as above, and200 mL substrate solution (1 OPD tablet/10 mL) was added. The OPD tabletcontains o-phenylenediamine dihydrochloride and hydrogen peroxide forhorse radish peroxidase reaction color development and is available fromSigma, St. Louis, Mo. This was incubated 30 minutes at 15-30° C. in thedark. The reaction was stopped by addition of 50 mL 4N H₂SO₄ and theplates were read at 492 nm, relative to absorbance at 690 nm as control.

D. Results:

The results of the various assays are shown in Tables 5 and 6, whichdepict two separate experiments done on blood samples exposed to HCVinfection as indicated. Shaded areas indicate detection of virus. Asshown in below, Chiron's combination antigen/antibody assay detectedseroconversion in all samples, while all other antibody- andantigen-based assays failed to detect seroconversion in at least onesample. In particular, neither of the antibody-based assays detectedseroconversion until at least day 18 (Table 5). Table 6 shows thatneither of the antibody-based assays detected the presence of HCVinfection at day 22. Moreover, the Ortho antigen-based assay failed todetect seroconversion from days 85 on.

Thus, based on the above results, it is clear that the novel combinationantibody/antigen assay reduces the number of false negatives obtainedusing other conventional antibody- and antigen-based assays.

TABLE 5 HCV Seroconversion

TABLE 6 HCV Seroconversion

Example 2 Production of an NS3/4a Conformational Epitope with Thr to Proand Ser to Ile Substitutions

A conformational epitope of NS3/4a was obtained as follows. This epitopehas the sequence specified in FIGS. 4A through 4D and differs from thenative sequence at positions 403 (amino acid 1428 of the HCV-1full-length sequence) and 404 (amino acid 1429 of the HCV-1 full-lengthsequence). Specifically, the Thr normally occurring at position 1428 ofthe native sequence has been mutated to Pro and Ser which occurs atposition 1429 of the native sequence has been mutated to Ile.

In particular, the yeast expression vector used was pBS24.1, describedabove. Plasmid pd.hcvla.ns3ns4aPI, which encoded a representative NS3/4aepitope used in the subject immunoassays, was produced as follows. A twostep procedure was used. First, the following DNA pieces were ligatedtogether: (a) synthetic oligonucleotides which would provide a 5′HindIII cloning site, followed by the sequence ACAAAACAAA, the initiatorATG, and codons for HCV1a, beginning with amino acid 1027 and continuingto a BglI site at amino acid 1046; (b) a 683 bp BglI-ClaI restrictionfragment (encoding amino acids 1046-1274) from pAcHLTns3ns4aPI; and (c)a pSP72 vector (Promega, Madison, Wis., GenBank/EMBL Accession NumberX65332) which had been digested with HindIII and ClaI, dephosphorylated,and gel-purified. Plasmid pAcHLTns3ns4aPI was derived from pAcHLT, abaculovirus expression vector commercially available from BD Pharmingen(San Diego, Calif.). In particular, a pAcHLT EcoRI-PstI vector wasprepared, as well as the following fragments: EcoRI-AlwnI, 935 bp,corresponding to amino acids 1027-1336 of the HCV-1 genome; AlwnI-SacII,247 bp, corresponding to amino acids 1336-1419 of the HCV-1 genome;HinfI-BglI, 175 bp, corresponding to amino acids 1449-1509 of the HCV-1genome; BglI-PstI, 619 bp, corresponding to amino acids 1510-1711 of theHCV-1 genome, plus the transcription termination codon. A SacII-HinfIsynthetically generated fragment of 91 bp, corresponding to amino acids1420-1448 of the HCV-1 genome and containing the PI mutations (Thr-1428mutated to Pro, Ser-1429 mutated to Ile), was ligated with the 175 bpHinfI-BglI fragment and the 619 bp BglI-PstI fragment described aboveand subcloned into a pGEM-5Zf(+) vector digested with SacII and PstI.pGEM-SZf(+) is a commercially available E. coli vector (Promega,Madison, Wis., GenBank/EMBL Accession Number X65308). Aftertransformation of competent HB101 cells, miniscreen analysis ofindividual clones and sequence verification, an 885 bp SacII-PstIfragment from pGEM5.PI clone2 was gel-purified. This fragment wasligated with the EcoRI-AlwnI 935 bp fragment, the AlwnI-SacII 247 bpfragment and the pAcHLT EcoRI-PstI vector, described above. Theresultant construct was named pAcHLTns3ns4aPI.

The ligation mixture above was transformed into HB101-competent cellsand plated on Luria agar plates containing 100 μg/ml ampicillin.Miniprep analyses of individual clones led to the identification ofputative positives, two of which were amplified. The plasmid DNA forpSP72 1aHC, clones #1 and #2 were prepared with a Qiagen Maxiprep kitand were sequenced.

Next, the following fragments were ligated together: (a) a 761 bpHindIII-ClaI fragment from pSP721aHC #1 (pSP72.1aHC was generated byligating together the following: pSP72 which had been digested withHindIII and ClaI, synthetic oligonucleotides which would provide a 5′HindIII cloning site, followed by the sequence ACAAAACAAA (SEQ ID NO:7),the initiation codon ATG, and codons for HCV1a, beginning with aminoacid 1027 and continuing to a BglII site at amino acid 1046, and a 683bp BglII-ClaI restriction fragment (encoding amino acids 1046-1274) frompAcHLTns3ns4aPI); (b) a 1353 bp BamHI-HindIII fragment for the yeasthybrid promoter ADH2/GAPDH; (c) a 1320 bp ClaI-SalI fragment (encodingHCV1a amino acids 1046-1711 with Thr 1428 mutated to Pro and Ser 1429mutated to Ile) from pAcHLTns3 ns4aPI; and (d) the pBS24.1 yeastexpression vector which had been digested with BamHI and SalI,dephosphorylated and gel-purified. The ligation mixture was transformedinto competent HB101 and plated on Luria agar plates containing 100μg/ml ampicillin. Miniprep analyses of individual colonies led to theidentification of clones with the expected 3446 bp BamHI-SalI insertwhich was comprised of the ADH2/GAPDH promoter, the initiator codon ATGand HCVLa NS3/4a from amino acids 1027-1711 (shown as amino acids 1-686of FIGS. 4A-4D), with Thr 1428 (amino acid position 403 of FIGS. 4A-4D)mutated to Pro and Ser 1429 (amino acid position 404 of FIGS. 4A-4D)mutated to Ile. The construct was named pd.HCV1a.ns3 ns4aPI (see, FIG.5).

S. cerevisiae strain AD3 was transformed with pd.HCV1a.ns3ns4aPI andsingle transformants were checked for expression after depletion ofglucose in the medium. The recombinant protein was expressed at highlevels in yeast, as detected by Coomassie blue staining and confirmed byimmunoblot analysis using a polyclonal antibody to the helicase domainof NS3.

Example 3 Purification of NS3/4a Conformational Epitope

The NS3/4a conformational epitope was purified as follows. S. cerevisiaecells from above, expressing the NS3/4a epitope were harvested asdescribed above. The cells were suspended in lysis buffer (50 mM Tris pH8.0, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin, 1 μMleupeptin) and lysed in a Dyno-Mill (Wab Willy A. Bachofon, Basel,Switzerland) or equivalent apparatus using glass beads, at a ratio of1:1:1 cells:buffer:0.5 mm glass beads. The lysate was centrifuged at30100×g for 30 min at 4° C. and the pellet containing the insolubleprotein fraction was added to wash buffer (6 ml/g start cell pelletweight) and rocked at room temperature for 15 min. The wash bufferconsisted of 50 mM NaPO₄ pH 8.0, 0.3 M NaCl, 5 mM β-mercaptoethanol, 10%glycerol, 0.05% octyl glucoside, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin,1 μM leupeptin. Cell debris was removed by centrifugation at 30100×g for30 min at 4° C. The supernatant was discarded and the pellet retained.

Protein was extracted from the pellet as follows. 6 ml/g extractionbuffer was added and rocked at room temperature for 15 min. Theextraction buffer consisted of 50 mM Tris pH 8.0, μM NaCl, 5 mMβ-mercaptoethanol, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 0.1 μM pepstatin,1 μM leupeptin. This was centrifuged at 30100×g for 30 min at 4° C. Thesupernatant was retained and ammonium sulfate added to 17.5% using thefollowing formula: volume of supernatant (ml) multiplied by x % ammoniumsulfate/(1−x % ammonium sulfate)=ml of 4.1 M saturated ammonium sulfateto add to the supernatant. The ammonium sulfate was added dropwise whilestirring on ice and the solution stirred on ice for 10 min. The solutionwas centrifuged at 17700×g for 30 min at 4° C. and the pellet retainedand stored at 2° C. to 8° C. for up to 48 hrs.

The pellet was resuspended and run on a Poly U column (Poly U Sepharose4B, Amersham Pharmacia) at 4° C. as follows. Pellet was resuspended in 6ml Poly U equilibration buffer per gram of pellet weight. Theequilibration buffer consisted of 25 mM HEPES pH 8.0, 200 mM NaCl, 5 mMDTT (added fresh), 10% glycerol, 1.2 octyl glucoside. The solution wasrocked at 4° C. for 15 min and centrifuged at 31000×g for 30 min at 4°C.

A Poly U column (1 ml resin per gram start pellet weight) was prepared.Linear flow rate was 60 cm/hr and packing flow rate was 133% of 60cm/hr. The column was equilibrated with equilibration buffer and thesupernatant of the resuspended ammonium sulfate pellet was loaded ontothe equilibrated column. The column was washed to baseline with theequilibration buffer and protein eluted with a step elution in thefollowing Poly U elution buffer: 25 mM HEPES pH 8.0, μM NaCl, 5 mM DTT(added fresh), 10% glycerol, 1.2 octyl glucoside. Column eluate was runon SDS-PAGE (Coomassie stained) and aliquots frozen and stored at −80°C. The presence of the NS3/4a epitope was confirmed by Western blot,using a polyclonal antibody directed against the NS3 protease domain anda monoclonal antibody against the 5-1-1 epitope (HCV 4a).

Additionally, protease enzyme activity was monitored during purificationas follows. An NS4A peptide (KKGSVIVGRIVLSGKPAIIPKK) (SEQ ID NO:8), andthe sample containing the NS3/4a conformational epitope, were diluted in90 μl of reaction buffer (25 mM Tris, pH 7.5, 0.15M NaCl, 0.5 mM EDTA,10% glycerol, 0.05 n-Dodecyl B-D-Maltoside, 5 mM DTT) and allowed to mixfor 30 minutes at room temperature. 90 μl of the mixture were added to amicrotiter plate (Costar, Inc., Corning, N.Y.) and 10 μl of HCVsubstrate (AnaSpec, Inc., San Jose Calif.) was added. The plate wasmixed and read on a Fluostar plate reader. Results were expressed asrelative fluorescence units (RFU) per minute.

Using these methods, the product of the 1 M NaCl extraction contained3.7 RFU/min activity, the ammonium sulfate precipitate had an activityof 7.5 RFU/min and the product of the Poly U purification had anactivity of 18.5 RFU/min.

Example 4 Competition Studies

The following competition study was conducted in order to assess whetherthe NS3/4a conformational epitope detected different antibodies thanother HCV antigens. In particular, the NS3/4a antigen was compared withthe c200 antigen as follows.

0.5 μg and 1.0 μg of NS3/4a, produced as described above, or c200(Hepatology (1992) 15:19-25, available in the ORTHO HCV Version 3.0ELISA Test System, Ortho-Clinical Diagnostics, Raritan, N.J.), weremixed with 20 μl of sample PHV914-5 (an early seroconversion bleedobtained from blood of an infected individual) in a total volume of 220μl (1×PBS). The mixture was incubated for 1 hour in microwells at 37° C.The mixture was then transferred to NS3/4a-coated plates and incubatedfor 1 hour at 37° C. Plates were washed and assayed as follows.

1 μg of c200 antigen was added to 10 μl of sample PHV914-5 in a totalvolume of about 220 μl. The mixture was incubated for 1 hour in a microwell at 37° C. and 200 μl transferred to an NS3/4a-coated plate (100ng/assay) and incubated for 1 hour at 37° C. Plates were washed fivetimes with 1×PBS, 0.1% Tween-20. 200 μl of conjugate solution (describedabove) were added, and the plates incubated and assayed. Controls whichconsisted of PHV914-5 and 1×PBS (without antigen) were also treated asabove.

Results are shown in Table 7. Percent inhibition results shown in column4 are calculated as column 3 minus (column 2 divided by column 3 times100). As can be seen, the data show that NS34a is neutralized by earlyseroconversion antibodies and c200 is not. A strong signal was achievedwhen antibodies in PHV914-5 c33c early seroconversion panel memberreacted with the NS34a coated on the plate. The c200 antigen was notneutralized by these antibodies. This is shown in the top panel of Table7. When NS34a was mixed with the PHV914-5 sample, it was neutralized andtherefore no antibodies were present in the sample to react with NS34athat was coated on the microplate. The data indicate that NS34a may bedetecting a different class of antibodies than is detected by c200.

TABLE 7 Competition Studies to Show NS34a Antigen Detects DifferentAntibodies in Early c33c Seroconversion Panel Compared to c200 Antigen

Example 5 Stability Studies of NS3/4a Conformational Epitope

To assess the role of stability of the NS3/4a epitope to assayperformance, the following study was done to determine NS3/4aimmunoreactivity versus time at room temperature. Small aliquots ofstock NS3/4a were allowed to sit at room temperature and then frozen atintervals as shown in Table 8. All vials were coated simultaneously andtested against two early NS3 seroconversion panels.

As can be seen in Table 8, the NS3/4a stock is not stable andimmunoreactivity decreases with time. In addition, maintaining NS3/4aconformation is necessary for immunoreactivity.

Further stability studies were conducted as follows. Two conformationalmonoclonal antibodies made against NS3/4a using standard procedures weresubstituted for anti-HCV early seroconversion panels. Stock NS3/4a vialswere stored at room temperature at time intervals 3, 6 and 24 hours. TheNS3/4a from the frozen vials was coated at 90 ng/ml and assayed usingthe procedure described above. Results suggested that the twomonoclonals were indeed conformational and their reactivity wassensitive to the handling of stock NS3/4a antigen at room temperature.The reactivity of a positive control monoclonal antibody did not change.

TABLE 8

Example 6 Immunoreactivity of NS3/4a Conformational Epitope VerusDenatured NS3/4a

The immunoreactivity of the NS3/4a conformational epitope, produced asdescribed above, was compared to NS3/4a which had been denatured byadding SDS to the NS3/4a conformational epitope preparation to a finalconcentration of 2%. The denatured NS3/4a and conformational NS3/4a werecoated onto microtiter plates as described above. The c200 antigen(Hepatology (1992) 15:19-25, available in the ORTHO HCV Version 3.0ELISA Test System, Ortho-Clinical Diagnostics, Raritan, N.J.) was alsocoated onto microtiter plates. The c200 antigen was used as a comparisonit is presumed to be non-conformational due to the presence of reducingagent (DTT) and detergent (SDS) in its formulation.

The immunoreactivity was tested against two early HCV seroconversionpanels, PHV 904 and PHV 914 (commercially available human blood samplesfrom Boston Biomedica, Inc., West Bridgewater, Mass.). The results areshown in Table 9. The data suggest that the denatured or linearized formof NS3/4a (as well as c200) does not detect early seroconversion panelsas early as the NS3/4a conformational epitope.

TABLE 9

Immunoreactivity of the conformational epitope was also tested usingmonoclonal antibodies to NS3/4a, made using standard procedures. Thesemonoclonal antibodies were then tested in the ELISA format againstNS3/4a and denatured NS3/4a and c200 antigen. The data show thatanti-NS3/4a monoclonals react to the NS3/4a and denatured NS3/4a in asimilar manner to the seroconversion panels shown in Table 10. Thisresult also provides further evidence that the NS3/4a is conformationalin nature as monoclonal antibodies can be made which are similar inreactivity to the early c33c seroconversion panels.

TABLE 10 Plate NS3/4a dNS3/4a c200 Monoclonal OD OD OD 4B9/E3 1:1001.820 0.616 0.369 1:1000 1.397 0.380 0.246 1:10000 0.864 0.173 0.0701:20000 0.607 0.116 0.085 5B7/D7 1:100 2.885 0.898 0.436 1:1000 2.8660.541 0.267 1:10000 1.672 0.215 0.086 1:20000 1.053 0.124 0.059 1A8/H21:100 1.020 0.169 0.080 1:1000 0.921 0.101 0.043 1:10000 0.653 0.0370.013 1:20000 0.337 0.027 0.011

Accordingly, novel HCV detection assays have been disclosed. From theforegoing, it will be appreciated that, although specific embodiments ofthe invention have been described herein for purposes of illustration,various modifications may be made without deviating from the spirit andscope of the disclosure herein.

1. A polynucleotide comprising a coding sequence for a multiple epitopefusion antigen consisting of the amino acid sequence of SEQ ID NO:5. 2.The polynucleotide of claim 1 comprising the nucleotide sequence of SEQID NO:4.
 3. A recombinant vector comprising: (a) a polynucleotideaccording to claim 1; (b) and control elements operably linked to saidpolynucleotide whereby the coding sequence can be transcribed andtranslated in a host cell.
 4. An isolated host cell transformed with therecombinant vector of claim
 3. 5. A method of producing a recombinantmultiple epitope fusion antigen comprising: (a) providing a populationof host cells according to claim 4; and (b) culturing said population ofcells under conditions whereby the multiple epitope fusion antigenencoded by the coding sequence present in said recombinant vector isexpressed.